A look at tricellulin and its role in tight junction formation and maintenance

Tight junctions are elaborate networks of transmembrane and cytosolic proteins that regulate epithelial permeability. Tricellulin was the first tight junction protein found at tricellular tight junctions, the specialized structures occurring where three cells meet together. Here, we summarize the current knowledge about tricellulin (marvelD2), a MARVEL domain protein. We address tricellulin location at tricellular junctions, and establish the comparison with the other members of the MARVEL family, occludin (marvelD1) and marvelD3. The structure of tricellulin and its membrane folding, as well as the proposed molecular interactions of tricellulin with other tight junction proteins, together with the interplay between those proteins are also discussed. In addition, we address the role of tricellulin in barrier properties, discriminating the involvement of the protein in paracellular permeability at bicellular and at tricellular tight junctions. Moreover, the key importance of the protein for hearing is highlighted based on the fact that mutations in TRIC, the human tricellulin gene, lead to deafness. Furthermore, this review points to some of the aspects that still deserve clarification for a better understanding of the biology of tight junctions in general and of tricellulin in particular.     

THE INTER-SERTOLI CELL TIGHT JUNCTIONS IN GERM CELL-FREE SEMINIFEROUS TUBULES FROM PRENATALLY IRRADIATED RATS: A FREEZE-FRACTURE STUDY

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood—testis barrier in the normal testis.     

Loss of occludin affects tricellular localization of tricellulin

The tricellular tight junction (tTJ) forms at the convergence of bicellular tight junctions (bTJs) where three epithelial cells meet in polarized epithelia, and it is required for the maintenance of the transepithelial barrier. Tricellulin is a four transmembrane domain protein recently identified as the first marker of tTJ, but little is known about how tricellulin is localized at tTJs. As for the molecular mechanism of association of tricellulin with tight junctions (TJs), we found that tricellulin was incorporated into claudin-based TJs independently of binding to zona occludens-1. Unexpectedly, exogenous expression of tricellulin increased cross-links of TJ strands in the plasma membrane. As for the molecular mechanisms for localization of tricellulin at tricellular junctions, we found that knockdown of occludin caused mislocalization of tricellulin to bTJs, implying that occludin supports tricellular localization of tricellulin by excluding tricellulin from bTJs.     

The gastric mucosal barrier: structure of intercellular junctions in the dog

The canine gastric mucosa consists of two regions, the surface mucous cells and gland area cells including parietal, chief, and mucous-containing cells. We have used quantitative freeze-fracture methods in conjunction with thin-section extracellular tracers to document and correlate tight junction morphology with epithelial permeability. The number of strands in the tight junction complexes of the surface cells and gland cells is the same, but differences in strand arrangement exist. The surface cells have an interwoven tight junction configuration which is impermeable to extracellular tracers. The gland cell junctions are regularly arranged and often permeable to extracellular lanthanum. The possibility that the observed difference in permeability between the tight junctions of the surface mucous cells and those of the gland cells is related to their structural configuration is discussed.     

Interactions of tight junctions with membrane channels and transporters

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.     

Interactions of tight junctions with membrane channels and transporters

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.     

c‐Jun N‐terminal kinase is largely involved in the regulation of tricellular tight junctions via tricellulin in human pancreatic duct epithelial cells

Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c‐Jun N‐terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), and the proinflammatory cytokines IL‐1β, TNFα, and IL‐1α. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL‐1β, TNFα, and IL‐1α, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT‐transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells. J. Cell. Physiol. 225: 720–733, 2010. © 2010 Wiley‐Liss, Inc.     

Comparison of the function of the tight junctions of endothelial cells and epithelial cells in regulating the movement of electrolytes and macromolecules across the cell monolayer

In cell culture, both endothelial and epithelial cell monolayers have been found to generate structurally similar tight junctional complexes, as assessed by thin complexes of the two cell types are, at least in part, responsible for the very different permeability characteristics of native endothelial and epithelial cell monolayers. The purpose of this work was to compare cultured endothelial and epithelial cells with respect to the function of their tight junctional complexes in regulating the movement of macromolecules and ions across the cell monolayers, and define functional parameters to characterize the tight junctional complexes. Bovine aorta endothelial cells and T84 colonic carcinoma epithelial cells were cultured on a microporous membrane support. The permeability coefficients of inulin, albumin, and insulin were determined with the cell monolayers and compared with the permeability coefficients obtained with 3T3-C2 fibroblasts, a cell line that does not generate tight junctions. Electrical resistance measurements across the monolayer-filter systems were also compared. The permeability coefficient of albumin across the endothelial cell monolayer compared favorably with other reported values. Likewise, the electrical resistance across the T84 cell monolayer was in good agreement with published values. Utilizing permeability coefficients for macromolecules as an index of tight junction function, we found that a distinction between a lack of tight junctions (fibroblasts), the presence of endothelial tight junctions, and the presence of epithelial tight junctions was readily made. However, when utilizing electrical resistance as an index of tight junction function, identical measurements were obtained with fibroblasts and endothelial cells. This indicates that more than one index of tight junction function is necessary to characterize the junctional complexes. Although structurally similar, epithelial cell and endothelial cell tight junctions perform very different functions, and, from our data, we conclude that the demonstration of tight junctional structures by electron microscopy is not relevant to the functional nature of the junction: structure does not imply function. A minimal assessment of tight junction function should rely on both the determination of the electrical resistance across the cell monolayer, and the determination of the permeability coefficients of selected macromolecules.     

Expression and distribution of symplekin regulates the assembly and function of the epithelial tight junction

Symplekin is multifunctional protein localized to both the tight junction and the nucleus with known roles in mRNA polyadenylation, proliferation, differentiation and tumorigenesis. Functions of symplekin at tight junctions have not been systematically investigated. In this study, increased expression of symplekin was observed during the formation of tight junctions in cultured HT-29 and HepG2 epithelial cells. Repression of symplekin by RNAi increased the permeability of epithelial monolayers, disrupted cellular polarity, and decreased the expression of the tight junction protein ZO-1. Moreover, symplekin was co-localized with ZO-1 at tight junctions and co-immunoprecipitated with ZO-1, indicating that ZO-1 and symplekin form complexes. In conclusion, symplekin expression regulates the assembly of tight junctions, thus helps to maintain the integrity of the epithelial monolayer and cellular polarity.     

Pubertal Development of Intersertoli Cell Junctions in the Testis of Corriedale Ram Lambs

The ultrastructure of the tight junctional complex of pubertal ram lamb Sertoli cells was studied in immersion-fixed samples and related to clinical data and a light microscopical classification of the degree of spermatogenesis attained in the corresponding seminiferous tubule. Although the process followed the general mammalian developmental trend for tight junction complex formation, 2 unusual ultrastructural features were detected: the presence of an active Golgi complex during the early stage of tight junction formation and the transitory presence of ribosomes on both faces of the ectoplasmic cisternae bordering the developing junctions. The significance of these findings is discussed.     

γ-Tubulin-like molecules in the mouse duodenal epithelium

A mouse monoclonal antibody (G9, Horio et al. in Cell Motil Cytoskel 44:284–295, 1999) that was raised against the γ-tubulin from a fission yeast, Schizosaccharomyces pombe, showed a unique staining in the mouse small intestine. Similar to another anti-γ-tubulin antibody that is commercially available, G9 showed typical dot-like staining corresponding to the microtubule-organizing center in the free cells of the epithelium and the connective tissue under it. In addition, G9 stained the cell–cell contacts in the epithelium. This stained region was not bicellular but tricellular junctions of the enterocytes. This staining was unique to G9 and was diminished on the sample of the mouse small intestine, which had lost most of its filamentous microtubules through the preparation process. The tricellular junction is thought to be the weakest point of the epithelial barrier, and no other junctional structures have been identified except for the central sealing elements extending from the tight junctions between the two cells. Our results suggest the existence of a new molecule underlying the tricellular junctions, which may relate to γ-tubulin and the microtubules.     

Regulation of tight junctions and loss of barrier function in pathophysiology

The mechanism by which epithelial and endothelial cells interact to form polarized tissue is of fundamental importance to multicellular organisms. Dysregulation of these barriers occurs in a variety of diseases, destroying the normal cellular environments and leading to organ failure. Increased levels of growth factors are a common characteristic of diseases exhibiting tissue permeability, suggesting that growth factors play a direct role in elevating permeability. Of particular concern for this laboratory, increased expression of vascular endothelial growth factor may enhance vascular permeability in diabetic retinopathy, leading to vision impairment and blindness. However, the mechanism by which growth factors increase permeability is unclear. Polarized cells form strong barriers through the development of tight junctions, which are specialized regions of the junctional complex. Tight junctions are composed of three types of transmembrane proteins, a number of peripheral membrane structural proteins, and are associated with a variety of regulatory proteins. Recent data suggest that growth factor-stimulated alterations in tight junctions contribute to permeability in a variety of disease states. The goal of this review was to elucidate potential mechanisms by which elevated growth factors elicit deregulated paracellular permeability via altered regulation of tight junctions, with particular emphasis on the tight junction proteins occludin and ZO-1, protein kinase C signaling, and endocytosis of junctional proteins. Understanding the molecular mechanisms underlying growth factor-mediated regulation of tight junctions will facilitate the development of novel treatments for diseases such as brain tumors, diabetic retinopathy and other diseases with compromised tight junction barriers.     

Na-K-ATPase regulates tight junction permeability through occludin phosphorylation in pancreatic epithelial cells

Tight junctions are crucial for maintaining the polarity and vectorial transport functions of epithelial cells. We and others have shown that Na-K-ATPase plays a key role in the organization and permeability of tight junctions in mammalian cells and analogous septate junctions in Drosophila. However, the mechanism by which Na-K-ATPase modulates tight junctions is not known. In this study, using a well-differentiated human pancreatic epithelial cell line HPAF-II, we demonstrate that Na-K-ATPase is present at the apical junctions and forms a complex with protein phosphatase-2A, a protein known to be present at tight junctions. Inhibition of Na-K-ATPase ion transport function reduced protein phosphatase-2A activity, hyperphosphorylated occludin, induced rearrangement of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na-K-ATPase is required for controlling the tight junction gate function.     

Shigella navigates tight corners

In this issue, Fukumatsu and colleagues (2012) find that Shigella preferentially spread from cell-to-cell at unique intercellular junctions. Shigella protrusions invade adjacent cells at junctions where three cells meet, the tricellular junction. The tight junction protein tricellulin marks these sites and is important for Shigella spread.     

Adjudin-mediated Sertoli–germ cell junction disassembly affects Sertoli cell barrier function in vitro and in vivo

Adjudin, an analogue of lonidamine, affects adhesion between Sertoli and most germ cells, resulting in reversible infertility in rats, rabbits and dogs. Previous studies have described the apical ectoplasmic specialization, a hybrid-type of Sertoli cell–elongating/elongated spermatid adhesive junction, as a key target of adjudin. In this study, we ask if the function of the blood–testis barrier which is constituted by co-existing tight junctions, desmosome-gap junctions and basal ectoplasmic specializations can be maintained when the seminiferous epithelium is under assault by adjudin. We report herein that administration of a single oral dose of adjudin to adult rats increased the levels of several tight junction and basal ectoplasmic specialization proteins during germ cell loss from the seminiferous epithelium. These findings were corroborated by a functional in vitro experiment when Sertoli cells were cultured on Matrigel?-coated bicameral units in the presence of adjudin and transepithelial electrical resistance was quantified across the epithelium. Indeed, the Sertoli cell permeability barrier was shown to become tighter after adjudin treatment as evidenced by an increase in transepithelial electrical resistance. Equally important, the blood–testis barrier in adjudin-treated rats was shown to be intact 2 weeks post-treatment when its integrity was monitored following vascular administration of inulin-fluorescein isothiocyanate which failed to permeate past the barrier and enter into the adluminal compartment. These results illustrate that a unique mechanism exists to maintain blood–testis barrier integrity at all costs, irrespective of the presence of germ cells in the seminiferous epithelium of the testis.     

Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells

For epithelia to function as barriers, the intercellular space must be sealed. Sealing two adjacent cells at bicellular tight junctions (bTJs) is well described with the discovery of the claudins. Yet, there are still barrier weak points at tricellular contacts, where three cells join together. In this study, we identify tricellulin, the first integral membrane protein that is concentrated at the vertically oriented TJ strands of tricellular contacts. When tricellulin expression was suppressed with RNA interference, the epithelial barrier was compromised, and tricellular contacts and bTJs were disorganized. These findings indicate the critical function of tricellulin for formation of the epithelial barrier.     

Tricellulin Forms a Barrier to Macromolecules in Tricellular Tight Junctions without Affecting Ion Permeability

Tricellulin is a tight junction protein localized in tricellular tight junctions (tTJs), the meeting points of three cells, but also in bicellular tight junctions (bTJs). To investigate its specific barrier functions in bTJs and tTJs, TRIC-a was expressed in low-level tricellulin–expressing cells, and MDCK II, either in all TJs or only in tTJs. When expressed in all TJs, tricellulin increased paracellular electrical resistance and decreased permeability to ions and larger solutes, which are associated with enhanced ultrastructural integrity of bTJs toward enhanced strand linearity. In tTJs in contrast, ultrastructure was unchanged and tricellulin minimized permeability to macromolecules but not to ions. This paradox is explained by properties of the tTJ central tube which is wide enough for passage of macromolecules, but too rare to contribute significantly to ion permeability. In conclusion, at low tricellulin expression the tTJ central tube forms a pathway for macromolecules. At higher expression, tricellulin forms a barrier in tTJs effective only for macromolecules and in bTJs for solutes of all sizes.     

Maturation of tight junctions in guinea-pig cecal epithelium

Summary By means of the freeze-fracture technique and in tracer studies it is demonstrated that the structure of tight junctions and the permeability to lanthanum of the guinea-pig cecal epithelium change during maturation of cells. Height and strand number of tight junctions in the apical-basal direction increase as crypt cells migrate to the surface of the epithelium. Likewise, the interlacing of continuous strands was greater in surface than in crypt junctions. The numerous free-ends, isolated individual freestrands and maculae occludentes found in crypt cells were absent in surface epithelial cells. Goblet cells, located at the bottom of crypts, displayed tight junctions similar in characteristics to those of cells located in the middle region of crypts. Cells at the surface and in middle regions of crypts possess tight junctions impermeable to lanthanum, whereas junctions between cells located at the bottom of crypts often were permeable to the tracer, indicating that permeability decreases as the epithelial cells mature. Genesis and maturation mechanisms related to structural configuration of tight junctions are discussed.     

Gliotactin,a novel marker of tricellular junctions,is necessary for septate junction development in Drosophila

Septate junctions (SJs), similar to tight junctions, function as transepithelial permeability barriers. Gliotactin (Gli) is a cholinesterase-like molecule that is necessary for blood-nerve barrier integrity, and may, therefore, contribute to SJ development or function. To address this hypothesis, we analyzed Gli expression and the Gli mutant phenotype in Drosophila epithelia. In Gli mutants, localization of SJ markers neurexin-IV, discs large, and coracle are disrupted. Furthermore, SJ barrier function is lost as determined by dye permeability assays. These data suggest that Gli is necessary for SJ formation. Surprisingly, Gli distribution only colocalizes with other SJ markers at tricellular junctions, suggesting that Gli has a unique function in SJ development. Ultrastructural analysis of Gli mutants supports this notion. In contrast to other SJ mutants in which septa are missing, septa are present in Gli mutants, but the junction has an immature morphology. We propose a model, whereby Gli acts at tricellular junctions to bind, anchor, or compact SJ strands apically during SJ development.     

Tight junction claudins and the kidney in sickness and in health

The epithelial cell tight junction has several functions including the control of paracellular transport between epithelial cells. Renal paracellular transport has been long recognized to exhibit unique characteristics within different segments of the nephron, functions as an important component of normal renal physiology and has been speculated to contribute to renal related pathology if functioning abnormally. The discovery of a large family of tight junction associated 4-transmembrane spanning domain proteins named claudins has advanced our understanding on how the paracellular permeability properties of tight junctions are determined. In the kidney, claudins are expressed in a nephron-specific pattern and are major determinants of the paracellular permeability of tight junctions in different nephron segments. The combination of nephron segment claudin expression patterns, inherited renal diseases, and renal epithelial cell culture models is providing important clues about how tight junction claudin molecules function in different segments of the nephron under normal and pathological conditions. This review discusses early observations of renal tubule paracellular transport and more recent information on the discovery of the claudin family of tight junction associated membrane proteins and how they relate to normal renal function as well as diseases of the human kidney.